PL241051B1 - Method of deposition of microorganisms or neoplastic cells on the SERS platform using the dielectrophoretic effect and marking microorganisms or neoplastic cells on the SERS platform - Google Patents

Abstract

The subject of the invention is a method of depositing tumor cells or bacteria on the SERS platform, comprising: preparing the SERS platform, introducing a suspension of bacteria, tumor cells in a carrier or yeast, transmitting an electrical signal to the SERS platform to deposit the bacteria or tumor cells on the platform, drying the SERS platform from with deposited bacteria or tumor cells, recording the SERS spectrum, characterized in that in step a) the SERS platform is placed in the measuring vessel and at least one SERS metal electrode is introduced into the measuring vessel, and in step c) the electrical signal is sent to at least one SERS metal electrode and the SERS platform to obtain a negative dielectrophoretic effect, and the electrical signal is transmitted to the metal SERS electrode and the SERS platform in 1 to 600 s, the electrical signal being at a frequency of 1 kHz to 50 MHz, the peak-to-peak voltage of the electrical signal being si from 1 Vpp to 50 Vpp.

Description

PL 241 051 B1

Description of the invention

The present invention relates to a method of depositing microorganisms or neoplastic cells by a negative dielectrophoretic effect for their determination by means of surface enhanced Raman spectroscopy on the SERS platform. The invention finds application in microbiological laboratories.

State of the art

The Surface Enhanced Raman Scattering (SERS) effect was discovered in the early 1970s by Fleischmann and colleagues who observed a strong amplification of the Raman signal for pyridine deposited on a rough electrode surface made of silver [1]. Interest in the SERS technique in life sciences is growing very quickly due to its non-destructive nature, speed of measurements and extremely high sensitivity, which allows the identification of substances with a concentration of ^10-12 mol / dm ³ , as well as individual DNA molecules, proteins, bacteria, viruses or circulating tumor cells in the blood [2, 3].

To observe the SERS effect, a metallic substrate is needed, most often gold and silver (also in the form of their alloys), which amplify the Raman signal. Moreover, the metal surface must have irregularities (corrugations, faults) or aggregates on the nanometer scale, most often between 10 nm and 100 nm [4, 5]. The signal gain ranges from 104 to 106, although gains greater than ^10–10 times have been reported [6, 7]. The size of the amplified SERS signal depends, among others, on from: frequency of excitation radiation (laser wavelength), effective Raman cross-section, type of analyte tested (chemical compound or biological material), electrical properties of the metal, distance of the molecule from the surface and surface morphology (size of crystallites and their geometry). The substrates used for SERS tests can be obtained, among others by electrochemical, lithographic, "template" methods or variations of chemical (CVD) and physical (PVD) vapor deposition methods [8, 9]. SERS test substrates can generally be divided into two main groups: nano-structured noble metal surfaces (Ag, Au as well as Cu, Pt, Pd) and nano-structures based on metal nanoparticles and colloids. It should be emphasized that to obtain the SERS platform, it is enough to cover the appropriate surface with a nanometer layer of gold or silver.

The following methods can be used to separate / overlay an appropriate number of microorganisms or neoplastic cells and aggregate them in one place on the SERS platform:

II) mechanical filtration: consisting in the use of membranes with appropriate pore sizes that allow the retention of microorganisms or neoplastic cells on their surface, and which can also be an active SERS substrate, as well as allow the discharge of unnecessary fluids [10],

III) magnetic separation and / or trapping: using magnetic nanoparticles which, thanks to appropriate chemical modification, are able to attach to bacteria or cancer cells. Gold or silver nanoparticles in suspension strengthen the Raman signal [11], or properly functionalized magnetic particles can simultaneously act as a separator and amplify the Raman signal [12],

IIII) chemical modifications of the surface and of the microorganisms themselves: with the use of polymer coatings, antibodies or other materials [13].

It is also possible to combine several methods of separation / deposition of microorganisms or neoplastic cells, eg modification of the substrate enabling the targeted capture of appropriate cells, and then applying them only to the tested SERS substrate [14, 15].

As already mentioned, dielectrophoresis can be a universal technique for detecting, analyzing, separating, fractionating or concentrating all kinds of materials, including biological materials. In relation to neoplastic cells, the possibility of using dielectrophoresis to assess the resistance of neoplastic cells to selected drugs [20], separating various types of neoplastic cells from each other, e.g. prostate and colon cancer [21], breast and colon [22] or catching circulating neoplastic cells was shown from blood plasma from patients [17, 23].

PL 241 051 B1

The American patent application US2009 / 0229980A1 presents a dielectrophoretic system with appropriately designed electrodes for the characterization and detection of suspended solids, mainly biological matter, including e.g. viruses, microorganisms (e.g. bacteria) and tumor cells in small volumes without the need for chemical tracers.

There are already works that try to link the deposition of bacterial or cancer cells using dielectrophoresis and then study them using Raman spectroscopy or surface-enhanced Raman spectroscopy.

At work, l.-F. Cheng et al. Presents a microfluidic system using the dielectrophoresis phenomenon with a rough metal surface made on glass for deposition / detection / identification of bacteria from blood samples using SERS. The roughened metal surface (Cr / Au) made on the glass was the SERS platform, and the sorting and concentration of bacteria was performed by means of three-dimensional dielectrophoresis. This allowed for a good amplification of the signal of the gram-positive and negative bacteria tested, as well as their identification [24].

In the work of H.-Y. Lin shows an integrated microfluidic device using the dielectrophoretic effect and a specially prepared bioconjugated SERS nanoprobe constituting a platform for the detection of bacteria. Thanks to the use of such a biosensor, it is possible to quickly identify even a single bacterium in the suspension [25].

In the work of U.-J. Schroder et al. Presented a microfluidic system combining the phenomenon of dielectophoresis and Raman spectroscopy. After pre-filtration of clinical specimens from UTI patients, the suspended patient specimen is injected into a microfluidic system where bacteria are trapped using dielectrophoretic force (which allows direct translational manipulation of bacteria in suspensions with spatial heterogeneous electric fields). Raman spectra are recorded directly from the suspension in places where bacteria clusters are formed. As shown in this paper, this configuration has the potential to shorten the diagnosis time of key parameters by orders of magnitude [26].

In the work of F.R. Madiyar et al. Presents a system for determining the concentration, detection and monitoring of pathogens, in particular bacteria, combining the phenomenon of dielectrophoresis with SERS nanotarkers that attached pathogens by immunochemistry. It allowed to record the spectrum of a single bacterium with the time of uptake of bacteria with DEP below 60 seconds [27].

The Polish patent application P.406900 describes "a polymer mat produced by the Forcespinning method, i.e. pulling a polymer fiber by centrifugal force, which was then covered with a layer of gold using the PVD method. This enabled the direct deposition of bacteria from the liquid, and the obtained SERS spectra were characterized by a high amplification of the Raman signal. On the other hand, the Polish patent PL232520B1 presents gold-sputtered polymer fiber mats, which constituted both the SERS platform, as well as a filter on which bacteria were deposited directly from the liquid (water, urine, plasma or apple juice) using negative pressure.

The main challenge with SERS substrates is to obtain a high enhancement factor (EF) while providing them with a homogeneous, repeatable structure at low cost of the production process itself, as well as its stability for a specified period of time (eg a month). In the case of the deposition of biological materials (e.g. bacteria, viruses, cancer cells), the problem is also the method of depositing / applying them to the test substrates, which in the case of biological materials would ensure their high density, thanks to which the obtained signal would be characterized by high amplification. Scientific publications and patents / patent applications do not provide information on the optimal method of applying bacteria to the substrate used in SERS measurements. In the case of clinical samples, another obstacle is the low concentration of microorganisms in body fluids, which makes it difficult to detect them directly in the suspension, therefore it is important to catch / separate and increase the density of the tested microorganisms or tumor cells from the solutions in which they are suspended (saline, plasma, blood, urine, cerebrospinal fluid and others). In addition, the known solution often requires the use of filters, trapping or chemical / biochemical modification of the surface of measuring vessels or microorganisms. Unexpectedly, the above problems were solved by the present invention.

The aim of the invention is to develop a method of deposition of microorganisms or neoplastic cells on the SERS platform using the negative dielectrophoretic effect (n-DEP) and the determination of microorganisms or neoplastic cells using these platforms.

PL 241 051 B1

The essence of the invention

The present invention relates to a method of depositing microorganisms or neoplastic cells on the SERS platform using the dielectrophoretic effect and determining the microorganisms or neoplastic cells on the SERS platform, comprising:

a) preparation of the SERS platform,

b) introducing the suspension of microorganisms or tumor cells in the carrier into the measuring vessel,

c) transmitting an electrical signal to the SERS platform and the SERS metal electrode in order to deposit microorganisms or neoplastic cells on the SERS platform, d) drying the SERS platform with microorganisms or neoplastic cells, e) recording the SERS spectrum, characterized in that in step a) the SERS platform is placed into the measurement vessel and at least one SERS metal electrode is introduced into the measurement vessel, and in step c) an electrical signal is transmitted to at least one SERS electrode and the SERS platform to obtain a negative dielectrophoretic effect, and the electrical signal to the metal electrode and SERS platform is transmitted for a period of 1 to 600 s, with the electrical signal frequency from 1 kHz to 50 MHz, with the peak-to-peak voltage of the electrical signal being from 1 Vpp to 50 Vpp.

In a preferred embodiment of the invention, the carrier is water, PBS buffer, a body fluid of human or animal origin selected from the group consisting of: blood, plasma, cerebrospinal fluid or urine.

In a further preferred embodiment of the invention, the peak-to-peak voltage is from 5 Vac to 30 Vac, most preferably from 10 Vac to 20 Vac.

In a further preferred embodiment of the invention, the electrical signal has a frequency more preferably in the range from 10 kHz to 20 MHz and most preferably in the range from 100 kHz to 10 MHz.

In yet another preferred embodiment of the invention, the electrical signal to the metal electrode SERS is transmitted in 10 s to 360 s, most preferably between 30 s to 60 s.

In a further preferred embodiment of the invention, the electrical signal to the metal electrode SERS is sent within 60 seconds.

In a further advantageous embodiment of the invention, the electrical signal transmitted to at least one SERS metal electrode and the SERS platform has a sinusoidal shape.

In another preferred embodiment, the diameter of the at least one SERS metal electrode is between 10 µm and 2 mm, more preferably between 20 µm and 1 mm, and most preferably between 50 µm and 500 µm. The smaller the diameter, the greater the electric field gradient, but the diameter cannot be reduced indefinitely, as a certain stiffness is required from the electrode to facilitate its assembly and increase its mechanical strength.

In another preferred embodiment of the invention, the diameter of the SERS metal electrode is 340 µm.

Preferably, after deposition of the microorganisms or tumor cells, the platform is removed and the carrier evaporated at room or elevated temperature. During deposition, the carrier in which the microorganisms or the neoplastic cells are embedded preferably covers the platform.

In another preferred embodiment of the invention, the measuring vessel is a cylindrical container made of plastic or glass, selected from the group consisting of: test tube, vial, sleeve.

In another preferred embodiment of the invention, the measuring vessel is a microfluidic system.

In a further preferred embodiment of the invention, the microfluidic system comprises two carrier liquid inlets connected to a mixer which are connected to a duct for separating tumor cells or bacteria from the carrier connected to the SERS platform chamber.

In a further preferred embodiment of the invention, four metal SERS electrodes are introduced into the SERS platform chamber of the microfluidic system, the metal SERS electrodes are introduced through channels in the SERS chamber perpendicular to each other, and the metal SERS electrodes are introduced above the SERS platform.

In another preferred embodiment of the invention, the distance between the ends of the metal electrodes inserted into the SERS platform chamber is not more than 1 mm, preferably 0.5 mm. The shorter the distance, the more efficiently cancer batteries / cells are deposited on the platform. At a distance of less than 0.5 mm between the electrodes, a short circuit may occur in the measuring circuit.

In a further advantageous embodiment of the invention, at least one SERS metal electrode is inserted parallel to at least one axis of the measuring vessel. The axis of the measuring vessel means, in the case of a cylindrical vessel, the axis corresponding to the axis of symmetry

PL 241 051 Β1 rotary measuring vessel. On the other hand, in the case where the measuring vessel is a microfluidic system, the axis of the measuring vessel can be understood as at least one axis parallel and coinciding with the channel for introducing the SERS metal electrode into the SERS platform chamber.

In yet another preferred embodiment of the invention, the SERS platform is a flexible dielectric barrier discharge foil, preferably of polyethylene terephthalate coated with a layer of indium tin oxide, to which a silver, gold or gold-silver alloy layer has been applied.

In a further preferred embodiment of the invention, the electrical signal is fed from an AC generator.

In yet another preferred embodiment of the invention, the neoplastic cells are selected from the group consisting of: K562 myeloid leukemia, HT-29 colorectal adenocarcinoma (0.1 Sm ^-1 ), BxPC3 pancreatic carcinoma, MCF7 breast cancer, HeLa cervical carcinoma, LNCaP prostate adenocarcinoma or MBA-MD-231 of breast cancer.

In another preferred embodiment of the invention, the microorganisms are selected from the group consisting of bacteria.

In a further preferred embodiment of the invention, the bacteria are gram-negative bacteria.

In yet another preferred embodiment of the invention, the gram-negative bacteria are selected from the group consisting of: Escherichia coli K.38, Escherichia coli 5K or Escherichia coli DoubleShell prolate.

In another preferred embodiment of the invention, the yeast is selected from the group consisting of: Saccharomyces cerevisiae or Saccharomyces cerevisiae.

In yet another preferred embodiment of the invention, the SERS platform is shaped to match the shape of the measurement vessel.

In another preferred embodiment of the invention, the SERS platform is L-shaped.

In order to deposit selected microorganisms or neoplastic cells, the invention uses the phenomenon of dielectrophoresis (DEP), which is a basic technology for the separation of molecules based on their manipulation in non-uniform electric fields.

The dielectrophoretic phenomenon was described in the 1950s by Pohl, who described the movement of molecules in a heterogeneous electric field [16]. Dielectrophoresis is the movement of objects caused by the application of a heterogeneous alternating electric field. Under the influence of this field, the object moves towards a greater density of electric field lines (positive dielectrophoresis, p-DEP) or towards an area with lower density of electric field lines (negative dielectrophoresis, n-DEP). Importantly, in the case of dielectrophoresis, unlike electrophoresis, the object does not have to have an electric charge on its surface. The phenomenon of dielectrophoresis can be described in more detail as follows. The stationary heterogeneous electric field E acting on a spherical particle placed in a liquid medium with a permittivity e _m causes the dielectrophoretic force, which is described by the equation (1):

^ dep = ^ ³ £ _m f _CM ^ \ E \, (1) where f _CM is the real part of the Claussius-Mossotti factor, which contains information about the dielectric properties, i.e. the permittivity and electrical conductivity of the medium and objects placed in it [17 ]:

where:

E - electric field strength (V / m), r - radius of the object (e.g. bacteria or cancer cell), ε _ρ - object electric permeability, e _s - electric permeability of the liquid in which the objects are suspended.

The first attempts to use this phenomenon for biological materials (yeast) for the segregation of living and dead cells were successfully made in the 1960s by Pohl [18]. In order to segregate different types of molecules, differences in their size as well as dielectric properties (conductivity and / or electric permeability) are used [19]. The process parameters, including the frequency of the alternating electric field, its intensity and the electrical conductivity of the medium in which the objects are suspended, should be properly selected.

PL 241 051 B1

The method according to the invention makes it possible to obtain a high density of the test material (cells, microorganisms) on the SERS medium, at its low concentration in the test sample, while the SERS medium itself provides a high amplification of the measured signal. Moreover, the solution according to the invention enables the deposition process to be carried out in a simple and low-cost manner, regardless of the type of biological sample. However, for deposition it is not necessary to use additional measures (filters, trapping or chemical / biochemical modification of the surface of the measuring vessels or microorganisms).

The invention will now be illustrated in more detail in the preferred embodiments with reference to the accompanying drawing, the elements being numbered to illustrate the implementation of the method according to the invention, so that the element having the same function in each embodiment has a similar reference, e.g. In an embodiment, the metal electrode is designated 1-2 and in the second embodiment, 2-2, in the third embodiment, the metal electrodes are designated 3-2, and so on.

Fig. 1 Plot of the real part of the Claussius-Mossotti factor (CM, expression 2.) versus the frequency of the alternating electric field. (a) Graph for HeLa tumor cell. A negative dielectrophoretic effect occurs at frequencies below 347 kHz; (b) a graph for Escherichia coli. A negative dielectrophoretic effect occurs at frequencies below 304 kHz. Calculations were made for HeLa tumor cells and E. coli bacteria placed in a 10-fold diluted aqueous solution of phosphate buffered saline (PBS) at pH 7.4. The diagrams show the frequency of the transition between negative DEP (n-DEP) and positive DEP (p-DEP) with a vertical dashed line;

Fig. 2 Schematic diagram of the advantageous use of the phenomenon of dielectrophoresis for the deposition of microorganisms or neoplastic cells in a cylindrical geometry. (a) A preferred embodiment of an L 1-1 shaped flexible electrode being SERS platform. In the lower part there is a DBD discharged PET / ITO film which is 1-1-1 SERS active. The upper part 1-1-2 is covered with a layer of conductive metal, which makes it possible to connect the signal from the function generator (1-6) to it. The width a is defined by the radius R of the cylinder 1-3 in which the electrodes are placed. (b) Use of the SERS platform to deposit bacteria or neoplastic cells. A metal electrode SERS 1-2 is inserted in the cylinder axis, with a diameter much smaller than that of the cylinder 1-3 used. Both electrodes: the flexible electrode, being the SERS 1-1 platform, and the metal electrode SERS 1-2, are connected to an alternating electric field source (functional generator, SIGLENT, SDG 2042X) 1-6. In the absence of the signal (OFF) (Fig. 2a), bacteria or tumor cells 1-4 are freely suspended in liquid 1-5. When the electric field is turned ON (Fig. 2b), the DEP force causes them to be deposited on the flexible electrode 1-1, namely on the active part SERS 1-1-1 located on the cylinder wall. (c) Electric field distribution in cylindrical geometry numerically calculated using the finite element method (Vpp = 20 V);

Fig. 3 Pictures of the experimental set-up in Fig. 2. (a) The function generator (SIGLENT, SDG 2042X) 1-6, which is the source of the alternating electric field, is connected to the experimental system in which there are two electrodes: metal electrode SERS 1-2 in the vial axis and the flexible electrode (PET / ITO / Ag foil) which is the SERS 1-1 platform. (b) Vial 1-3 filled with a liquid containing bacteria. Function generator 1-6 is connected to the electrodes via the electrical terminals;

Fig. 4 Averaged spectra of LNCaP cells (prostate adenocarcinoma derived from metastases to the left supraclavicular lymph node of a 50-year-old male), obtained on a flexible PET / ITO film subjected to a dielectric barrier discharge and then coated with a silver layer with a thickness of 70 nm, for the example of the first preferred an embodiment of the invention cylindrical geometry; LNCAp - a line of human prostate adenocarcinoma cells derived from metastases to the left supraclavicular lymph node of a 50-year-old male (collected in 1977);

Fig. 5 Schematic diagram of the advantageous use of the phenomenon of dielectrophoresis for the deposition of microorganisms or neoplastic cells on the SERS platform with an area limited by a cylinder 2-3, a hollow with a height of several mm. (a) The substrate is a PET / ITO / Ag 2-1 film, and a sharp SERS 2-2 metal electrode is used as the second electrode. Both electrodes receive an electrical signal from function generator 2-6. When the tension is on

In the OFF position (Fig. 5), in the area delimited by the walls of the cylinder 2-3, there are freely suspended bacteria 2-4 or tumor cells 2-4. When the voltage is turned ON (Figure 5), the force of DEP causes the bacteria or tumor cells to move to the surface of the SERS 2-1 platform. (b) The electric field distribution between the pointed metal electrode SERS 2-2 and the platform SERS 2-1 calculated numerically using the finite element method (Vpp = 20V);

Fig. 6 (a) Photo of the experimental setup shown in Fig. 5. (b) Close-up of the SERS platform

2-1, a cylinder limiting the outflow of liquid (2-3) and a pointed metal electrode SERS 2-2. The alternating electrical signal is fed through the electrical terminals from Function Generator 2-6. (c) Scanning Electron Microscope (SEM) image of the end of the SERS 2-2 metal electrode;

Fig. 7 Averaged SERS spectra of HeLa cervical cancer cells obtained on a flexible PET / ITO film subjected to a dielectric barrier discharge and then coated with a 70 nm silver layer for an example of a second preferred embodiment of the invention SERS platform with constrained geometry;

Fig. 8 Photo of the microflow system including: 3-3 - measuring chamber, 3-7 - channels for metal electrodes (dielectrophoretic deposition of bacteria or tumor cells on the surface of the SERS platform), 3-8 - inlets for liquid administration, 3-9 - mixer microfluidic, 3-10 - channels for preliminary metal electrodes (preliminary separation of bacteria or cancer cells using the DEP effect), 3-11 - liquid outlet from the microflow system, 3-12 - liquid outlet from the measuring chamber, 3-13 - connecting channel preliminary separation channel with a measuring chamber, 3-14 - channel for preliminary separation of bacteria or neoplastic cells using the DEP effect);

Fig. 9 Schematic diagram of the advantageous use of the phenomenon of dielectrophoresis for deposition of microorganisms or neoplastic cells from the dielectophoretic trap. (a) The trap consists of a polycarbonate plate with a milled chamber 3-3 and four channels 3-7 leading to it metal electrodes 3-2. At the bottom of the chamber there is a SERS 3-1 platform (PET / ITO / Ag foil), above it are four pointed metal electrodes SERS 3-2 (see Fig. 5b). The signal is supplied to the electrodes from two function generators 3-6 or from one generator having two independent channels. (b) Cross-section through an array showing chamber 3-3 with the SERS platform 3-1 and metal electrodes 3-2 located above the SERS platform. The distance between the metal electrode and the SERS platform should be 0.5 mm or more. (c) Due to an alternating electric field of appropriate frequency, the bacteria or tumor cells are subjected to a dielectrophoretic force (n-DEP) directed at the area between the pointed electrodes 3-2, where bacteria / tumor cells accumulate 3-4. (d) The distribution of the electric field between the electrodes calculated numerically using the finite element method (Vpp = 20 V);

Fig. 10 (a) Photo of the experimental set-up including the microfluidic circuit of Fig. 9, metal electrodes, and electrical terminals running to the output of the function generator. (b) The fragment of the microfluidic system of Fig. 8 comprises a channel 3-13 in which a liquid flows with suspended bacteria or tumor cells. It flows into chamber 3-3, to which four channels 3-7 are connected for the introduction of metal electrodes 3-1. (c) For the measurement, a SERS 3-1 platform in the form of a PET / ITO foil coated with a silver layer is placed on the bottom of the chamber 3-3, and metal electrodes SERS 3-2 are inserted through the channels. A solution containing bacteria or cancer cells flows into the chamber, and it flows out through the outlet (3-12). When the alternating electric field is turned on, the bacteria accumulate on the surface of the SERS 3-1 platform between the ends of the electrodes (see Fig. 9c) due to the action of the dielectrophoretic force;

Fig. 11 Averaged spectra of Escherichia coli gram (-) bacteria obtained on a flexible PET / ITO film subjected to a dielectric barrier discharge and then coated with a silver layer with a thickness of 70 nm for an example of a third preferred embodiment of the invention a four electrode array (dielectrophoretic trap).

Example 1: cylindrical geometry

The method of preparing flexible SERS platforms based on 125 μm PET foil, covered with a 5 μm ITO layer, subjected to a dielectric barrier discharge (DBD) and covered with a thin metal layer (silver, gold, copper, silver coated with a layer of gold, silver and gold alloy)

PL 241 051 Β1 or gold plated copper). The SERS platform is the subject of the Polish patent application No. P.430767.

The method of deposition of microorganisms or neoplastic cells is based on the use of the SERS platform in the form of a flexible PET / ITO-based foil covered with a silver layer, enabling it to be placed in a cylindrical container (glass vial or test tube mat). After applying an alternating electric field between the SERS platform and the second electrode in the axis of the container, the objects are affected by a negative dielectrophoretic effect (n-DEP), i.e. a dielectrophoretic force directed towards the SERS platform. It causes the bacteria or cancer cells to move towards the SERS platform and, consequently, to settle them there. The frequency of the transition (/ transition), where the / transition is Re [CM (f)] = 0, is calculated numerically using the dependence of the real part of the Claussius-Mossotti factor on the frequency of the applied alternating electric field (Fig. 1). An exemplary Claussius-Mossotti factor plot for a HeLa tumor cell is shown in Figure 1a. The frequency of the transition between the negative DEP effect and the positive DEP effect for HeLa cells is 347 kHz. To obtain a negative dielectrophoretic effect (n-DEP) in the system, we must use a frequency lower than 347 kHz. In the case of LNCaP neoplastic cells, the frequency of 1 MHz ensured the n-DEP effect. The frequency of the transition was taken from the literature, reference [21]. Exemplary calculated transition frequencies for various microorganisms are shown in the tables below.

Table 1

Tumor line crossing rates calculated for x10 diluted PBS, calculated with myDEP software

Cancer lines Crossover / Crossover frequency {kHz) K562 myeloid leukemia 716 HT-29 colorectal adenocarcinoma (0.1 Sm ¹ ) 897 BxPC3 pancreatic cancer cells 182 MCF7 breast cancer cells 197 HeLa cervical cancer cells 348 LNCaP of prostate adenocarcinoma cells 20,000 * MBA-MD-231 breast cancer cells 228

* the transition frequency for the LNCaP cell line was taken from reference [21]

Table 2

Bacterial transition frequencies based on x10 diluted PBS, calculated with myDEP software

Bacteria The frequency of the transition / transition (^ Hz) Escherichia coli K. 38 305 Escherichia coli 5K 1590 Escherichia coli (DoubleShell prolate) 1130

PL 241 051 Β1

Table 3

Crossover frequencies for yeast calculated with x10 diluted PBS, calculated with myDEP software

Yeast The frequency of the transition / transition (kHz) Saccharomyces cerevisiae 4790 Saccharomyces cerevisiae expressing 5te2p 4250

The SERS platform is L 1-1 shaped as shown in Fig. 2a. The upper part of the PET / ITO 1-1-2 film is one of the electrodes to which the electric field is applied, while the lower part 1-1-1 is the actual SERS platform onto which the microorganisms / tumor cells will be deposited. The deposition process of bacteria or neoplastic cells is schematically shown in Fig. 2b. The length of the lower part of the platform a is calculated from the radius R of the cylindrical container (a = 2kR). The second electrode (metal SERS electrode) in the form of a thin steel wire (1-2) is placed along the axis of the cylindrical vessel. A large electric field gradient is created between the electrodes, which is required in the dielectrophoresis process. The numerically calculated electric field distribution is shown in Fig. 2c.

LNCaP tumor cells were suspended in 10-fold diluted PBS with a conductivity of 156 mS m ^-1 . A long side L-shaped PET / ITO film of 30.0 mm χ 3.0 mm was inserted into a cylindrical glass test tube with a diameter of 9.5 mm. The shorter side, which was the actual SERS platform, was 7.0 mm χ 7.0 mm. 250 µl of a 10-fold diluted aqueous PBS solution containing the tumor cells was introduced into the test tube. The volume of the liquid was chosen to completely cover the SERS platform.

In order to deposit LNCaP tumor cells on the SERS platform, a variable electrical signal was applied between the electrode being the SERS platform and the steel wire electrode in the axis of the tube. The steel electrode may have a diameter of 10 µm to 2 mm, more preferably the diameter is 20 µm to 1 mm and most preferably 50 µm to 500 µm. A steel electrode with a diameter of 340 µm was used in the experiment. Sinusoidal signal with a frequency of 1 MHz (value calculated numerically for specific physicochemical values of LNCaP cancer cells and the electrical conductivity of the buffer) and a peak-to-peak voltage of 10 V _PP . The optimal deposition time (experimentally selected) was 60 seconds. The experimental setup together with the Siglent SDG 2042Χ (1-6) functional generator used to pellet LNCaP tumor cells is shown in Fig. 3.

After 60 seconds, the platform was removed from the tube, dried at room temperature or elevated temperature (T> 30 ° C) and subjected to recording of the SERS spectrum. In order to carry out the measurements, a Bruker Bravo Raman spectrometer equipped with the Duo Laser ™ technology was used. The laser wavelength was between 700 nm and 1100 nm and the power was below 100 mW. Spectra were recorded with 3 6000 ms accumulations each and averaged.

Table 4

Effect of deposition time on the intensity of Raman signals for E. coli bacteria and LNCaP tumor cell

Tested systems Deposition time (seconds) The intensity of the diagnostic band 731 cm ¹ for E. coli and 650 cm ¹ for LNCaP (number of counts) Escherichia coli K. 38 thirty 18500 60 15400 180 10200 LNCaP of prostate adenocarcinoma cells thirty 12,000 60 8500 180 4700

PL 241 051 B1

The averaged spectrum of LNCaP tumor cells deposited on the SERS platform by the dielectrophoretic effect (n-DEP) is shown in Fig. 4.

Example 2: SERS platform with limited geometry

In the case of small-volume samples containing bacteria or neoplastic cells, another method of their deposition using the dielectrophoretic effect was proposed.

The method is based on the use of PET / ITO / Ag foil to which a plastic sleeve with a diameter of 5 mm and a wall thickness of 1 mm is attached to the side of the silver layer. The sleeve is sealed from the outside with a small amount of two-component adhesive. Inside the sleeve, a liquid with suspended bacteria or tumor cells 2-4 is introduced, and from the top, in the sleeve axis, a pointed metal electrode, SERS 2-2, is introduced. The electrode must be inserted into the liquid but must not touch the silver layer. The schematic diagram of the presented solution is shown in Fig. 5. The function generator 2-6 is connected to the lower electrode (SERS platform) 2-1 and the pointed metal electrode SERS 2-2. When the generator output is not active (Fig. 5a, OFF), bacteria or tumor cells are freely suspended in the liquid. When the generator output is active (Fig. 5a, ON), there is a high gradient alternating electric field between the SERS platform 2-1 and the pointed metal electrode SERS 2-2 (SEM photo shown in Fig. 6c). The pointed metal electrode is responsible for this gradient (the electric field distribution calculated numerically using the finite element method is shown in Fig. 5b). As a result, bacteria or cancer cells are subjected to a dielectrophoretic force directed towards the SERS platform (n-DEP), causing the movement and deposition of objects on the surface of the platform.

In the experiment, a stop sleeve 2-3 with a diameter of 5 mm was attached to a flexible SERS platform with dimensions of 7.0 mm χ 7.0 mm. 70 μl of a 10-fold diluted PBS solution containing HeLa tumor cells was loaded into the sleeve. The experimental setup is shown in Figs. 6a and 6b. The conductivity of the solution was 156 mSm ^-1 . A sinusoidal signal with a frequency of 300 kHz and a peak-to-peak voltage of 10 Vpp were used to deposit HeLa tumor cells using the n-DEP effect. The optimal deposition time (determined experimentally) was 60 seconds.

The frequency at which n-DEP occurs was determined from the plot of the real part of the Claussius-Mossotti factor (CM) versus the frequency of the applied alternating current acting on the HeLa cell placed in an aqueous solution of phosphate buffered saline pH = 7.4 (PBS, diluted χ10) . This diagram is shown in Fig. 1a. It shows the frequency of the transition between negative DEP (n-DEP) and positive DEP (p-DEP), which for this system is 347 kHz. The use of the 300 kHz frequency therefore ensures that n-DEP acts on the objects, directing them to the SERS platform.

After 60 seconds, the platform was dried and subjected to recording of the SERS spectrum. In order to carry out the measurements, a Bruker Bravo Raman spectrometer equipped with the Duo Laser ™ technology was used. The laser wavelength was between 700 nm and 1100 nm and the power was below 100 mW. Spectra were recorded with 3 6000 ms accumulations each and averaged.

The averaged spectrum of HeLa tumor cells mounted on the SERS platform by the dielectrophoretic effect (n-DEP) is shown in Fig. 7.

The proposed method can be applied to any SERS platform: based on silicon, photovoltaic panels, membranes etc.

For a material that will be hydrophobic, it will not be necessary to have liquid-confining walls as this will form a spherical droplet.

Example 3: Four electrode system - Dielectrophoretic trap

The dielectrophoretic effect is an attractive mechanism for separating bacteria or cancer cells from water, urine, blood or plasma, and is therefore often used in conjunction with microfluidic and (microfluidic) techniques.

In this example, we demonstrate a microfluidic system (Fig. 8), which includes: a measuring chamber 3-3, a channel for metal electrodes 3-7 for the dielectrophoretic deposition of bacteria or tumor cells on the surface of the SERS platform, liquid inlets 3-8, a mixer microfluidic 3-9, channel for metal electrodes 3-10 for the initial separation of bacteria or cancer cells using the DEP effect, liquid outlet from the microflow system

3-11, the liquid outlet from the measuring chamber 3-12, the channel connecting the preliminary separation channel with the measuring chamber 3-13, the channel for the initial separation of bacteria or tumor cells by the DEP effect 3-14.

In the side walls of the measuring chamber 3-3 there are four channels 3-7 for inserting metal electrodes SERS 3-2. Metal electrodes SERS 3-2 having sharp ends (Fig. 6c) are arranged radially to each other and at right angles to the adjacent electrode, with a gap between the ends of the electrodes of approx. 1 mm. The electrodes 3-2 are located above the surface of the platform SERS 3-1 with their ends exactly in the center of the platform and chamber 3-3 (Fig. 9a and Fig. 9b). The electrodes 3-2 are connected to the outputs of the function generator 3-6; each pair of electrodes 3-2 to a separate channel as shown in Fig. 9a. After switching on an alternating electric field of an appropriate frequency, the bacteria are subjected to a dielectrophoretic force (n-DEP) directed towards the space between the electrodes (Fig. 9c). The distribution of the electric field is shown in Fig. 9d. The geometry of the electrodes, and thus the distribution of the electric field, causes the objects to be subjected to a negative dielectrophoretic force, accumulating bacteria or tumor cells in the center, exactly between the electrode tips (Figs. 9c, 3-4). This is called dielectrophoretic trap. In our case, the SERS platform is located under the electrodes, thanks to which objects (cancer cells or bacteria) are deposited on it in one place.

The microfluidic system milled in polycarbonate with connected tubing and electrodes is shown in Fig. 10a. The size of the chamber shown in Fig. 10b is: 5.0mm × 5.0mm × 2.5mm (3-3). The SERS platform is a PET / ITO / Ag 3-1 film with dimensions of 4.5 mm χ 4.5 mm. The electrodes 3-2 were inserted so that the distance between their ends was no more than 1 mm (Fig. 9b and Fig. 9c). They are also located above the surface of the SERS 3-1 platform, their height is determined by the appropriate diameter of the electrodes 3-2 and the location of the channels in the side walls of the chamber 3-7 (Fig. 9b).

The PBS solution (diluted χ 10) containing Escherichia coli bacteria was applied to the prepared system. A sinusoidal signal frequency of 200 kHz and a peak-to-peak voltage of 15 Vpp were used to deposit E. coli on the SERS platform. The deposition time was 60 seconds.

After 60 seconds, the platform was removed from the chamber, dried and subjected to recording of the SERS spectrum. In order to carry out the measurements, a Bruker Bravo Raman spectrometer equipped with the Duo Laser ™ technology was used. The laser wavelength was between 700 nm and 1100 nm, and the power was below 100 mW. Spectra were recorded with 3 6000 ms accumulations each and averaged.

The averaged spectrum of Escherichia coli bacteria deposited on the SERS platform by means of the dielectrophoretic effect (n-DEP) is shown in Fig. 11.

The proposed method can be applied to any SERS platform: based on silicon, photovoltaic panels, membranes etc. In the case of SERS platforms with a thickness greater than PET / ITO foil, the height of the channels for metal electrodes should be changed (the height should be selected experimentally so that there is no short circuit between the electrodes).

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Claims (25)

PL 241 051 B1 Patent claims 1. A method of depositing microorganisms or neoplastic cells on the SERS platform using a dielectrophoretic effect and marking microorganisms or neoplastic cells on the SERS platform, comprising: a) preparation of the SERS platform, b) introducing the suspension of microorganisms or tumor cells in the carrier into the measuring vessel, c) transmitting an electrical signal to the SERS platform and a metal electrode in order to deposit microorganisms or neoplastic cells on the SERS platform, d) drying the SERS platform with microorganisms or cancer cells, e) recording the SERS spectrum for the determination of microorganisms or tumor cells, characterized in that in step a) the SERS platform is placed in the measurement vessel and at least one metal SERS electrode is inserted into the measurement vessel, and in step c) an electrical signal is transmitted to at least one the metal SERS electrode and the SERS platform to obtain a negative dielectrophoretic effect, and the electrical signal to the metal SERS electrode and the SERS platform is transmitted in 1 to 600 s, the electrical signal being at a frequency of 1 kHz to 50 MHz, with the peak-to-peak voltage the electrical signal is from 1 Vpp to 50 Vpp. 2. The method according to p. The process of claim 1, wherein the carrier is water, PBS buffer, a body fluid of human or animal origin selected from the group consisting of: blood, plasma, cerebrospinal fluid or urine. 3. The method according to p. The method of claim 1, wherein the peak-to-peak voltage of the electrical signal is from 5 Vac to 30 Vac, most preferably from 10 Vac to 20 Vac. 4. The method according to p. The electric signal according to claim 1 or 3, characterized in that the electric signal has a frequency in the range from 10 kHz to 20 MHz, most preferably in the range from 100 kHz to 10 MHz. 5. The method according to p. 1, 3 or 4, characterized in that the electric signal to the metal electrode SERS is transmitted during 10 s to 360 s, most preferably between 30 s to 60 s. 6. The method according to p. 1, 3, 4 or 5, characterized in that the electrical signal to the metal electrode SERS is transmitted during 60 s. 7. The method according to p. A method as claimed in any one of claims 1, 3, 4, 5 or 6, characterized in that the electrical signal transmitted to at least one SERS metal electrode and the SERS platform has a sinusoidal shape. 8. The method according to p. The method of claim 1, characterized in that the diameter of at least one SERS metal electrode has a diameter of 10 µm to 2 mm, more preferably 20 µm to 1 mm, most preferably 50 µm to 500 µm. 9. The method according to p. 8. The method of claim 8, characterized in that the diameter of the SERS metal electrode is 340 Pm. 10. The method according to p. The method of claim 1, wherein after pelleting of the microorganisms or tumor cells, the SERS platform is removed and the carrier is evaporated at room or elevated temperature. 11. The method according to p. The method of claim 1, characterized in that the measuring vessel is a cylindrical plastic or glass container selected from the group consisting of: test tube, vial, sleeve. 12. The method according to p. The method of claim 1, characterized in that the measuring vessel is a microfluidic system. 13. The method according to p. The method of claim 12, characterized in that the microfluidic system comprises two carrier liquid inlets (3-8) connected to a mixer (3-9) which are connected to a channel for pre-separation of tumor cells or bacteria from the carrier (3-14) connected by the carrier liquid outlet (3-10) through the channel (3-13) with the SERS platform chamber (3-3), the SERS platform placement chamber (3-3) including channels (3-7) for the introduction of SERS metal electrodes (3 -2). 14. The method according to p. 12 or 13, characterized in that four metal SERS electrodes (3-2) are inserted into the SERS platform chamber (3-3), while the metal SERS electrodes (3-2) are introduced through the channels (3-7) in the SERS platform chamber (3-3) perpendicular to each other, and the metal SERS electrodes (3-2) are inserted above the SERS platform (3-1) in the SERS platform chamber (3-1). PL 241 051 B1 15. The method according to p. 14. The method of claim 14, characterized in that the distance between the ends of the SERS metal electrodes (3-2) inserted into the SERS platform chamber (3-3) is not more than 1 mm, preferably 0.5 mm. 16. The method according to p. The method of claim 1, characterized in that at least one SERS metal electrode is inserted parallel to at least one axis of the measuring vessel. 17. The method according to p. The method of claim 1, characterized in that the SERS platform is a flexible dielectric barrier discharge film, preferably polyethylene terephthalate coated with an indium tin oxide layer onto which a silver, gold or gold-silver alloy layer has been applied. 18. The method according to p. The method of claim 1, 3, 4, 5, 6 or 7, characterized in that the electrical signal is supplied from an alternating current generator. 19. The method according to p. The method of claim 1, characterized in that the neoplastic cells are selected from the group consisting of: K562 myeloid leukemia, HT-29 colorectal adenocarcinoma (0.1 Sm ^-1 ), BxPC3 pancreatic cancer, MCF7 breast cancer, HeLa cervical cancer, LNCaP prostate adenocarcinoma or MBA-MD-231 of breast cancer. 20. The method according to p. The process of claim 1, wherein the microorganisms are selected from the group consisting of bacteria or yeasts. 21. The method according to p. The method of claim 20, characterized in that the bacteria are gram-negative bacteria. 22. The method according to p. The method of claim 21, wherein the gram-negative bacteria are selected from the group consisting of: Escherichia coli K.38, Escherichia coli 5K or Escherichia coli DoubleShell prolate. 23. The method according to p. The process of claim 20, wherein the yeast is selected from the group consisting of: Saccharomyces cerevisiae or Saccharomyces cerevisiae. 24. The method according to p. The method of claim 1, characterized in that the SERS platform has a shape adapted to the shape of the measurement vessel. 25. The method according to p. 24, characterized in that the SERS platform is L-shaped.